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[ Chest Infections Research Letter ] 56

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broker. Sections were stained with human anti-IL-33
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IL-33 Depletion in (R&D Systems, AF3625, Lot #YYZ0918051, 2.5 mg/100
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8 COVID-19 Lungs mL), prosurfactant protein C (Millipore Sigma, AB3786, 63
9 Lot #3466551, 1:200), and vimentin (Abcam, AB 92547, 64
To the Editor:
10 Lot #GR3258719-9, 1:200). Donkey anti-goat 65
11 IL-33 is an alarmin that plays an integral role in lung (AlexaFluorPlus555, A32816, Lot #VB299353) and anti- 66
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homeostasis through its actions in wound repair, rabbit (AlexaFluor488, A21206, Lot #2156521) from
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fibrosis, and remodeling processes.1 Stored in the Thermo Fisher at 1:100 dilutions were used.
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nucleus, IL-33 is released to the cytoplasm and Photographs (10 per lung section per patient) of lung
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extracellular fluids following insult or damage that was parenchyma were taken from the entire section under a
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induced by various infectious, noxious, or Zeiss fluorescent microscope (Carl Zeiss Microscopy) Q5 72
18 environmental agents.2 In addition to its role in allergic at 20 magnification. A total of 11 COVID-19 sections 73
19 asthma,3,4 studies have demonstrated elevated IL-33 in (each patient with one section, except three patients had 74
20 COPD plasma,5 COPD airways,1 and idiopathic two sections each, representing different regions of the 75
21 pulmonary fibrosis (IPF) lung tissues6; however, a lung) were included. The integrated densities (the 76
22 comparative analysis of lung IL-33 expression in the product of area and mean gray value) of each protein 77
23 setting of infectious sequalae are lacking. Infection with were measured as single color on black background with 78
24 SARS-CoV-2 causes an inflammatory cascade that color threshold by Image J software (version: 2.1.0/ 79
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results in reduced diffusion capacity, hypoxia, and 1.53c). Statistical analysis was conducted with averaged
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Q1 death.7,8 The REACT COVID investigators screened densities of each patient with Prism 9 software (version:
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serum from 100 subjects with COVID-19 for cytokines 9.0.0) with the use of the Mann-Whitney test vs control
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Q2 (ie, IL-6, tumor necrosis factor, IL-8, IL-1ß, granulocyte- group; a probability value of <.05 was accepted as
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Q3 macrophage colony-stimulating factor, IL-33, statistically significant.
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Q4 interferon-ɣ, IL-10) and found that increased serum IL-
31 Tissue IL-33 expression was increased significantly in 86
32 33 levels (as well as tumor necrosis factor) were 87
IPF (6.57-fold; P ¼ .0012) and COPD (3.91-fold;
33 independently predictive of poor outcomes with SARS- 88
P ¼ .0012) compared with control subjects, whereas
34 CoV-2 in patients <70 years old (adjusted OR for IL-33, 89
patients with COVID-19 had low to negligible IL-33
35 11.14; 95% CI, 1.01-123.72).9 The objective of this study 90
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expression that was significantly reduced as compared
was to characterize IL-33 expression in the lungs of 91
37 with control subjects (0.03-fold; P ¼ .0003) (Fig 1A-C). 92
patients with fulminant COVID-19, comparing this
38 Costaining with prosurfactant protein C was used to 93
expression with that observed in other inflammatory
39 assess type II alveolar epithelial cells (AEC2); vimentin 94
lung diseases.
40 stain was used to assess mesenchymal cells (ie, 95
41 De-identified, postmortem lung sections of patients with fibroblasts, smooth muscle cells, and endothelial cells) 96
42 COVID-19 (N ¼ 8; age, 35 to 85 years; female, 25%; and macrophages (Fig 1A-B). In control subjects, IL-33 97
43 smokers, 50%; hypertensive, 87.5%; obese, 50%; diabetic, expression was predominately nuclear and localized to 98
44 62.5%; vascular disease, 50%) were obtained from the endothelial cells. In comparison, patients with IPF and 99
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University of Nebraska Medical Center institutional COPD demonstrated increased nuclear and
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review board-approved lung and/or cardiology bio- cytoplasmic IL-33 expression in endothelial cells,
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banks. De-identified samples from normal human lungs macrophages, and AEC2. Minimal expression of IL-33
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(“controls”) deemed unsuitable for transplantation (N ¼ was demonstrated in COVID-19 lungs in some
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7; age range, 19 to 59 years; female, 14.3%; smokers, vimentinþ endothelial cells. Vimentin staining was 105
51 57%), IPF (N ¼ 4; age range, 47 to 69 years; female, increased in COVID-19 (2.15-fold; P ¼ .0093) and IPF 106
52 25%), COPD (N ¼ 6; age range, 57 to 64 years; female, (1.74-fold; P ¼ .0424) as compared with control 107
53 50%), and post-COVID fibrosis (N ¼ 1) with limited subjects with no difference between COPD and controls 108
54 clinical information were obtained from explanted lungs (Fig 1D). As compared with control subjects, AEC2 109
55 from the lung transplant bio-bank through an honest numbers were decreased in COVID-19 (0.01-fold; 110

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111 166
A Vimentin IL-33 DAPI Merge
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114 Control 169
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117 IPF 172
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121 COPD 176
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124 COVID 179
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127 Post- 182
128 COVID 183
129 Fibrosis 184
130 185
131 B proSP-C IL-33 DAPI Merge 186
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133 Control 188
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IPF
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140 COPD 195
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COVID
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145 200
146 201
Post-
147 COVID 202
148 fibrosis 203
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150 C D E 205
151 4 × 105 3 × 105 206
Integrated Density of Vimentin

Integrated Density of proSP-C

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2× 106
Integrated Density of IL-33

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3 × 105
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2× 105
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1 × 106
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1× 105
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3 × 104
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5 × 105 212
158 4 × 103 1 × 104 ### 213
159 2 × 103 5 × 103 214
print & web 4C=FPO

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os ID

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os D

C D
ID
C F
PD
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ID

l
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tro
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C D

os D
C F

ID
IP
IP

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br VI

tro
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on

Fi CO
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165 Figure 1 – Lung expression of IL-33, vimentin and prosurfactant protein C among healthy control subjects, idiopathic pulmonary fibrosis , COPD, 220
COVID, and post-COVID-19 fibrosis. Photomicrographs (10 per lung section per patient) were taken of the entire lung section under a (Continued)

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221 P ¼ .0003) and COPD (0.43-fold; P ¼ .0047) lungs with underscore the complexity of IL-33 in lung disease 276
222 no difference between IPF and control subjects (Fig because lung IL-33 expression was increased strikingly 277
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1E). In post-COVID fibrosis, IL-33, vimentin, and in chronic lung disease, whereby serum IL-33 levels can
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AEC2 were all increased to levels at or above that also be elevated variably.5,6 IL-33 is a key mediator not
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demonstrated in COPD and IPF (Fig 1A-E). only in danger signaling but also in wound repair and
226 281
lung recovery processes that can be marked by
227 Lung tissue IL-33 was expressed in basal epithelial cells, 282
dysregulated fibrosis. Investigating IL-33 levels in the
228 AEC2, endothelial cells, fibroblasts, macrophages, and 283
229 lung of survivors of COVID-19 would also provide 284
other progenitor cells with markedly increased
230 insight into restoration of IL-33 in normal homeostasis. 285
expression in COPD and IPF. However, IL-33 was
231 Indeed, IL-33 and AEC2 expression was increased in 286
nearly entirely depleted from the lung tissue of subjects
232 post-COVID fibrosis lung to support these future 287
with COVID with negligible availability or reserve in the
233 studies. Whether replenishment of IL-33 by lung 288
nucleus of any lung cell. Five of eight COVID-19 lungs
234 progenitor cells as observed in chronic disease states 289
235
demonstrated extracellular or cytoplasmic IL-33 290
would be beneficial or harmful in the setting of
236 expression, but at extremely low levels to suggest release 291
overwhelming infection is not known. In contrast,
237 and depletion. These findings also corresponded to a 292
blocking viral-mediated IL-33 release early in the
238 near absence of prosurfactant protein Cþ AEC2 in the 293
infectious process could be explored. When an integral
239 COVID-19 lungs potentially to suggest cellular death 294
role of IL-33 in Th2 diseases is considered, future studies
240 and/or lack of progenitor epithelial cells to aid in lung 295
241
could also use asthmatic lung samples in comparison 296
repair and recovery processes. There was wider patient
242 studies. A limitation of this study is that IL-33 protein 297
variability in vimentin expression with COVID-19,
243 expression was assessed by immunohistochemistry 298
which could reflect variation in time course of COVID
244 because of availability; IL-33 investigations in various 299
infection (timing unknown). Similar to COVID-19,9
245 compartments that include lavage fluid, tissue, and 300
serum IL-33 levels are also increased with influenza and
246 serum at both protein and gene expression level to 301
lung IL-33 increases in healthy control subjects who
247 inform the role of IL-33 fully in SARS-CoV-2 are 302
248
were infected with influenza.10 However, there remains a 303
warranted.
249 gap of knowledge as to whether other fulminant 304
250 infectious respiratory diseases are also associated with In conclusion, these studies strengthen the relationship 305
251 IL-33 exhaustion. of IL-33 in COVID-19 to suggest that additional and 306
252 longitudinal assessments are warranted to understand 307
Corticosteroids are used commonly in COVID-19 and
253 the mechanisms and timing of lung IL-33 expression 308
254
can down-regulate several cytokines,11 but IL-33 has 309
and regulation for promoting damage or driving wound
255 been recognized to be nonresponsive to glucocorticoid 310
repair processes to inform potential interventional
256 therapy.3,4 Moreover, all bio-banked lungs received 311
strategies.
257 glucocorticoids in the standard optimization procedure 312
258 before harvest. It remains possible that circulating IL-33- 313
259 producing cells are also important in the response to Rohit Gaurav, PhD Q18 314
260 SARS-CoV-2 infection; however, the striking depletion Daniel R. Anderson, MD, PhD 315
261 of lung tissue IL-33 suggests the importance of a lung Stanley J. Radio, MD 316
262 compartment-specific source of IL-33. It is also noted Kristina L. Bailey, MD 317
263 318
that IL-33 is cleaved to a number of inflammatory Bryant R. England, MD, PhD
264 319
products that potentially were not detected; however, the Ted R. Mikuls, MD, MSPH
265 320
antibody that was used recognizes mature and cleaved Geoffrey M. Thiele, PhD
266 321
267
forms through recognition of the Ser112-Thr270 amino Heather M. Strah, MD
322
268 acid sequence of IL-33. Nonetheless, these studies Debra J. Romberger, MD 323
269 324
270 Figure 1 | (Continued) Zeiss fluorescent microscope (Carl Zeiss Microscopy) at 20 magnification. Representative images are shown with areas of Q16 325
271 interest boxed in yellow. A, Vimentin (green), IL-33 (red), and DAPI (blue) stains and a merge image with all three stains are shown for each 326
investigated group. B, Prosurfactant protein C (green), IL-33 (red), DAPI (blue) stains and a merge image with all three stains are shown for each Q19
272 327
investigated group. Scatter plots depict median of averaged integrated densities per each patient for IL-33. C, IL-33. D, Vimentin. E, Prosurfactant
273 protein C . The individual values are averaged from ten images per section per patient for the respective proteins. Because IL-33 was stained with both 328
274 vimentin and prosurfactant protein C, 20 images (ten from vimentin and ten from prosurfactant protein C costaining) are from each subject. Statistical 329
difference is denoted in the following manner: #P < .05; ##P < .01; ###P < .0001. IPF ¼ idiopathic pulmonary fibrosis; proSP-C ¼ prosurfactant
275 330
protein C.

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331 Todd A. Wyatt, PhD B.R. England, T.R. Mikuls, G.M. Thiele, H.M. Strah, D.J. Romberger, 386
332 T.A. Wyatt, J.D. Dickinson, M.J. Duryee, D.M. Katafiasz, A.J. Nelson, 387
John D. Dickinson, MD, PhD
J.A. Poole: conception of the work; R. Gaurav, K.L. Bailey, J.A. Poole:
333 Michael J. Duryee, MS design of the work; R. Gaurav, S.J. Radio, J.A. Poole: acquisition, 388
334 Dawn M. Katafiasz, MPH analysis, & interpretation of data for the work; R. Gaurav, J.A. Poole: 389
335 drafted the manuscript; D.R. Anderson, D.J. Romberger, J.D. 390
Amy J. Nelson, MT Dickinson, M.J. Duryee, D.M. Katafiasz, A.J. Nelson: acquisition of
336 Q6 data for the work; R. Gaurav, D.R. Anderson, S.J. Radio, K.L. Bailey, 391
Jill A. Poole, MD
337 B.R. England, T.R. Mikuls, G.M. Thiele, H.M. Strah, D.J. Romberger, 392
Omaha, NE T.A. Wyatt, J.D. Dickinson, M.J. Duryee, D.M. Katafiasz, A.J. Nelson:
338 393
performed critical analysis and revision of the draft; R. Gaurav, D.R.
339 AFFILIATIONS: From the Division of Allergy & Immunology (R. Anderson, S.J. Radio, K.L. Bailey, B.R. England, T.R. Mikuls, G.M. 394
340 Gaurav, A.J. Nelson, and J.A. Poole), the Departments of Thiele, H.M. Strah, D.J. Romberger, T.A. Wyatt, J.D. Dickinson, M.J. 395
341 Cardiovascular Medicine (D.R. Anderson), Pathology & Duryee, D.M. Katafiasz, A.J. Nelson, J.A. Poole: approved the 396
Microbiology (S.J. Radio), Pulmonary Critical Care & Sleep (K.L. manuscript and are accountable for all aspects of the work.
342 Bailey, H.M. Strah, D.J. Romberger, T.A. Wyatt, J.D. Dickinson, and 397
343 D.M. Katafiasz), Rheumatology & Immunology, Department of Other contributors: The authors wish to thank Lisa Chudomelka for Q11
398
Internal Medicine (B.R. England, T.R. Mikuls, G.M. Thiele, and M.J. assistance with manuscript preparation/submission.
344 399
Duryee), University of Nebraska Medical Center (UNMC); the VA
345 Nebraska Western Iowa Health Care System (K.L. Bailey, B.R. 400
Q7
346 England, T.R. Mikuls, D.J. Romberger, and T.A. Wyatt); and References 401
347 Environmental, Agricultural & Occupational Health (T.A. Wyatt), 1. Byers DE, Alexander-Brett J, Patel AC, et al. Long-term IL-33- 402
UNMC. producing epithelial progenitor cells in chronic obstructive lung
348 Q8 Q12 Q13
403
FUNDING/SUPPORT: R. Gaurav is supported by Central States disease. J Clin Invest. 2013;123:3967-3982.
349 Center for Agricultural Safety and Health NIOSH (U54 OH010162). 404
2. Gaurav R, Varasteh JT, Weaver MR, et al. The R213G
350 Intramural UNMC COVID-19 grant (D.R. Anderson), National polymorphism in SOD3 protects against allergic airway 405
Institute of Environmental Health Sciences (R01ES019325 to J.A. inflammation. JCI Insight. 2017;2. Q14
351 406
Poole), National Institute for Occupational Safety and Health
352 (U54OH010162 to J.A. Poole). T.R. Mikuls is supported by a VA 3. Saglani S, Lui S, Ullmann N, et al. IL-33 promotes airway 407
remodeling in pediatric patients with severe steroid-resistant
353 Merit Award (CS000896) and grants from the National Institute of
asthma. J Allergy Clin Immunol. 2013;132:676-685. 408
General Medical Sciences (U54GM115458) and the National
354 4. Gordon ED, Simpson LJ, Rios CL, et al. Alternative splicing of 409
Institute on Alcohol Abuse and Alcoholism (R25AA020818).
355 National Institute for Occupational Safety and Health interleukin-33 and type 2 inflammation in asthma. Proc Natl Acad 410
356 (U54OH010162 to J.A. Poole and T.A. Wyatt). T.A. Wyatt is the Sci U S A. 2016;113:8765-8770. 411
recipient of a Research Career Scientist Award (IK6BX003781) from 5. Kim SW, Rhee CK, Kim KU, et al. Factors associated with plasma
357 412
the Department of Veterans Affairs. B.R. England is supported by IL-33 levels in patients with chronic obstructive pulmonary disease.
358 grants from the National Institute of General Medical Sciences Int J Chron Obstruct Pulmon Dis. 2017;12:395-402. 413
359 (U54GM115458) and the Rheumatology Research Foundation. The 6. Luzina IG, Pickering EM, Kopach P, et al. Full-length IL-33 414
study was also supported by the Fred & Pamela Buffett Cancer
360 Q17
promotes inflammation but not Th2 response in vivo in an ST2- 415
Center Tissue Sciences Facility) Shared Resource, supported by the independent fashion. J Immunol. 2012;189:403-410.
361 National Cancer Institute under award number P30CA036727. 416
362 7. Gattinoni L, Coppola S, Cressoni M, Busana M, Rossi S, 417
This publication’s contents are the sole responsibility of the authors. Chiumello D. COVID-19 does not lead to a “typical” acute
363 The funders had no role in study design, data collection and analysis, respiratory distress syndrome. Am J Respir Crit Care Med. 2020;201: 418
decision to publish, or preparation of the manuscript. 1299-1300.
364 419
Q9 FINANCIAL/NONFINANCIAL DISCLOSURES: None declared.
365 8. Hue S, Beldi-Ferchiou A, Bendib I, et al. Uncontrolled innate and 420
Q10 CORRESPONDENCE TO: Jill A. Poole, MD; email: japoole@unmc.edu impaired adaptive immune responses in patients with COVID-19
366 Copyright Ó 2021 Published by Elsevier Inc under license from the ARDS. Am J Respir Crit Care Med. Forthcoming. Q15 421
367 American College of Chest Physicians. 9. Burke H, Freeman A, Cellura DC, et al. Inflammatory phenotyping 422
368 DOI: https://doi.org/10.1016/j.chest.2021.06.058 predicts clinical outcome in COVID-19. Respir Res. 2020;21:245. 423
369 10. Hawley RG, Covarrubias L, Hawley T, Mintz B. Handicapped 424
370
Acknowledgments retroviral vectors efficiently transduce foreign genes into 425
Author contributions: Jill A. Poole takes responsibility for (is the hematopoietic stem cells. Proc Natl Acad Sci U S A. 1987;84:2406-
371 2410. 426
guarantor of) the content of the manuscript, including the data and
372 analysis. The following authors are responsible for the various aspects 11. Barnes PJ. How corticosteroids control inflammation: Quintiles 427
373 of the manuscript, as indicated: R. Gaurav, D.R. Anderson, S.J. Radio, Prize Lecture 2005. Br J Pharmacol. 2006;148:245-254. 428
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